Regulation of pacemaker neurons The molecular mechanisms driving structural and functional plasticity in the brain are key for human health but not very well understood in any model system. Circadian pacemaker neurons are among the best understood central brain neurons, studied at multiple levels from genes to neural circuits. They show 24hr rhythms in neuronal activity and in structural and synaptic plasticity, programmed by their intrinsic molecular clocks. Thus pacemaker neurons offer an unusual opportunity to understand the interplay between gene expression, neuronal activity and plasticity. We identified a Rho GTPase GEF (Pura) whose rhythmic expression drives rhythms in Rho1 activity in the s-LNv principal pacemaker neurons in Drosophila. This in turn drives rhythms in the structure and synaptic composition of s-LNv axonal termini. We propose to build on these data using pacemaker neurons to understand the regulation and function of neuronal plasticity. Pura is the fly orthologue of human Puratrophin- 1, which is mutated in spinocerebellar ataxia. In Aim 1 we propose to identify the mechanism of Pura regulation. In contrast to typical activity- dependent genes, Pura is repressed by neuronal activity and activated by hyperpolarization. We have generated a Pura transcriptional reporter that will allow us to identify the relevant DNA response elements and screen for transcription factors that mediate this response in LNvs. We will also test if hyperpolarization- dependent gene expression is a general phenomenon by generating additional reporters for genes co- regulated with Pura in LNvs and by testing if Pura responds to hyperpolarization in other neurons. In Aim 2, we propose to study the function of Pura and Rho1 in pacemaker neuron plasticity. Pura and Rho1 regulate s-LNv morphology and the numbers of active zones and levels of dendritic markers, which we hypothesize are mediated by distinct Rho1 effectors. We will manipulate individual Rho1 effectors with spatial and temporal specificity to determine their individual contributions to s-LNv plasticity. This should give molecular insights into the mechanisms of synapse assembly and disassembly. We will also measure the effect of s-LNv plasticity on communication with downstream neurons. In Aim 3, we propose to identify additional GEFs and Rho family GTPases that contribute to LNv plasticity. Our LNv expression profiles identified two additional GEFs that are either rhythmically expressed or clock-controlled in LNvs, but with opposite regulation to Pura. We hypothesize that these GEFs regulate Rac1 and/or Cdc42 GTPases to help s-LNv projections expand at dawn.